Compositions and methods for treating and preventing inflammation
The present invention provides novel compounds compositions and methods for (i) treating or preventing inflammation; and (ii) preventing or reducing hyperactivation of innate immune response, by inhibiting NRP1-dependent cell-signaling. Also provided are compounds, composition, and methods of specifically inhibiting SEMA3A-mediated cell signaling.
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This is a continuation of U.S. application Ser. No. 16/422,273, filed May 24, 2019, issued as U.S. Pat. No. 10,766,964 on Sep. 8, 2020, which is a divisional of U.S. application Ser. No. 15/507,407, filed Feb. 28, 2017, issued as U.S. Pat. No. 10,738,122 on Aug. 11, 2020, which is the U.S. National Stage of International Patent Application No. PCT/CA2015/050862, filed Sep. 8, 2015, which was published in English under PCT Article 21(2), which in turn claims priority of U.S. provisional application serial No. U.S. 62/046,459, filed on Sep. 5, 2014. The above-referenced applications are incorporated herein by reference in their entirety.
SEQUENCE LISTINGThis application contains a Sequence Listing in computer readable form entitled “98565-03_ST25.txt”, created on Jul. 30, 2020 having a size of 579 KB. The computer readable form is incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTN.A.
FIELD OF THE INVENTIONThe present invention relates to inflammation. More specifically, the present invention is concerned with the inhibition of the NRP1 pathway for the prevention or treatment of inflammation.
REFERENCE TO SEQUENCE LISTINGN.A.
BACKGROUND OF THE INVENTIONLocal acute inflammatory responses are predominantly beneficial and constitute the body's first line of defense against infection of the host. Conversely, acute systemic inflammation such as in septic shock is a leading cause of morbidity and mortality (58). When chronic, low-grade inflammation persists, it can be at the origin of a several systemic diseases ranging from type II Diabetes Mellitus, arthritis, cancer, a number of neuro-inflammatory conditions and more.
Of all cytokines, receptors and other players thought to contribute to the inflammatory processes, one paradigm that has been largely overlooked is the influence of classical neuronal guidance cues and their receptors. These include semaphorin3A (SEMA3A, e.g., mRNA: NM_006080; and protein: NP_006071 and
The Semaphorins were initially characterized as key players in axonal guidance during embryogenesis. It is now clear that the role of Semaphorins extends beyond axonal guidance and influence vascular systems, tumor growth and the immune response. The Semaphorin family counts at least 21 vertebrate genes and 8 additional genes in invertebrates. All Semaphorins contain a ˜500 amino acid SEMA domain that is required for signaling. Class 3 Semaphorins (such as SEMA3A) are the only secreted members of the family. SEMA3A is synthesized as a disulphide-linked homodimer and dimerization is essential for signaling.
In neurons, binding of SEMA3A to its cognate receptor Neuropilin-1 (NRP1) provokes cytoskeletal collapse via plexins (60); the transduction mechanism in endothelial cells remains ill-defined. NRP1 has the particular ability to bind two structurally dissimilar ligands via distinct sites on its extracellular domain (27-29). It binds not only SEMA3A (46, 47) provoking cytoskeletal collapse but also VEGF165 (28, 29, 47, 61) enhancing binding to VEGFR2 and thus increasing its angiogenic potential (62). Crystallographic evidence revealed that VEGF165 and SEMA3A do not directly compete for NRP1 but rather can simultaneously bind to NRP1 at distinct, non-overlapping sites (63). Moreover, genetic studies show that NRP1 distinctly regulates the effects of VEGF and SEMA3A on neuronal and vascular development (64). Finally, NRP1 has also been found to bind to TGF-β1 and to regulate its latent form.
NRP1 is a single-pass transmembrane receptor with a large 860 amino acid extracellular domain subdivided into 3 sub-domains (a1a2, b1b2 and c) and a short 40 amino acid intracellular domain (65). In neurons, binding of SEMA3A to NRP1 recruits Plexins, which transduce their intracellular signal (60) and provoke cytoskeletal collapse. The transduction mechanism in endothelial cells remains ill-defined. NRP1 binds SEMA3A (46, 47) primarily via its a1a2 (but possibly also b1-) domain (provoking cytoskeletal collapse) and VEGF165 (28, 29, 47, 61) via its b1b2 domain (enhancing binding to VEGFR2 and thus increasing its angiogenic potential (62). The elevated levels of SEMA3A in the ischemic retina may thus partake in forcing neovessels into the vitreous by collapsing and deviating the advancing tip cells away from the source of the repellent cue (21).
The CNS had long been considered an immune-privileged system, yet it is now clear that the brain, retina and spinal cord are subjected to complex immune-surveillance (1, 2). Immunological activity in the CNS is largely dependent on an innate immune response and is present in health and heightened in diseases such as diabetic retinopathy, multiple sclerosis, amyotrophic lateral sclerosis and Alzheimer's disease. This is apparent in the retina where an intensified, largely microglial/macrophage-based immune response is associated with the progression of several sight threatening diseases such as diabetic retinopathy (DR)(3-5), age related macular degeneration (AMD)(6-8) and retinopathy of prematurity (ROP)(9, 10). Together, these retinal diseases account for the principal causes of loss of sight in industrialized countries (6, 11, 12).
Many of the current line of treatments of inflammatory diseases and conditions suffer from important side-effects and deficient long-term safety profiles. Accordingly, there remains a need for novel pharmaceutical targets and methods of treatments.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTIONThe present inventors have sought to determine the function of myeloid-resident NRP1 in the context of the innate immune response.
The present inventors have determined that SEMA3A, VEGF and TGF-β act as potent attractants for mononuclear phagocytes (MPs, e.g., microglia and macrophages) expressing the NRP1 receptor. Inhibition of NRP1 signaling in innate immune cells was shown to result in protection against MPs-dependent inflammation and tissue damage under a variety of conditions involving hyperactivation of the innate immune-response including proliferative retinopathies, septic shock and cerebral ischemia/stroke. Furthermore, the present inventors have designed various soluble NRP1-derived traps which inhibit SEMA3A signalling and shown that inhibition of SEMA3A significantly reduce the inflammatory response in various conditions.
Accordingly, the present invention relates to the inhibition of NRP1 cell signalling (e.g., NRP1 and its ligands) for the prevention or treatment of inflammatory diseases and conditions involving hyperactivation (i.e., pathological activation) of the innate immune response. Non-limiting examples of such disease and conditions include sepsis, stroke, cerebral ischemia, and various proliferative retinopathies.
More specifically, in an aspect, the present invention concerns a method of treating or preventing inflammation comprising inhibiting NRP1-dependent cell-signaling.
In another aspect, the present invention relates to a method of preventing or reducing hyperactivation of innate immune response comprising inhibiting NRP1-dependent cell-signaling. In an embodiment, the hyperactivation of innate immune response comprises i) secretion of IL-1β and TNFα and/or activation/recruitment of mononuclear phagocytes (MPs).
In an embodiment, inhibiting NRP1-dependent cell-signaling comprises: a) reducing NRP1 expression or activity; and/or b) reducing NRP1 ligand expression or activity. In an embodiment, the NRP1 ligand is SEMA3A, VEGF165 or TGF-β. In a particular embodiment, the NRP1 ligand is SEMA3A.
In an embodiment, reducing NRP1 activity consists of inhibiting the binding of NRP1 to at least one NRP1 ligand. In an embodiment, inhibiting the binding of NRP1 to at least one NRP1 ligand comprises administering an NRP1 antibody (e.g., a SEMA3A antibody).
In another embodiment of the above methods, reducing NRP1 activity comprises administering an effective amount of an NRP1 trap which comprises soluble NRP1 polypeptide or a functional fragment thereof. In a particular embodiment, the NRP1 trap is as set forth in
In a particular embodiment, the NRP1 trap of the present invention inhibits the binding of SEMA3A to NPR-1 but does not substantially inhibit the binding of VEGF to NRP1. In an embodiment, such NRP1 trap comprises the a1a2 domain of NRP1 but does not comprise the b1 and/or b2 subdomain(s) of NRP1. In another embodiment, such trap comprises a mutation in domain b1 at a position corresponding to tyrosine 297 of the NRP1 amino acid sequence as set forth in
In specific embodiments, the NRP1 trap of the present invention: a) comprises domains a1, a2, b1, b2 and c and of NRP1; b) comprises domains a1, a2, b1 and b2 of NRP1; c) comprises domains a1, a2 and b1 of NRP1; d) comprises domains a1 and a2 of NRP1; e) comprises domain b1, wherein the b1 domain comprises a mutation in amino acid corresponding to tyrosine 297 of NRP1 which reduces or abrogates the binding to VEGF; f) comprises domain b1, wherein the b1 domain comprises a mutation in amino acid corresponding to tyrosine 297 of NRP1 which changes the tyrosine to an alanine; g) does not comprise domain c of NRP1; h) does not comprise domain b1 of NRP1; i) does not comprise domains b1 and b2 of NRP1; or j) does not comprise domains b1, b2 and c of NRP1.
In an embodiment of the above methods, inhibiting NRP1 ligand expression or activity comprises specifically inhibiting SEMA3A expression or SEMA3A binding to NRP1. In a particular embodiment, inhibiting SEMA3A binding to NRP1 comprises administering a SEMA3A antibody.
In a particular embodiment, the method of the present invention comprises reducing NRP1 expression by administering a NRP1 antisense, shRNA or siRNA.
In another embodiment, the method comprises reducing SEMA3A expression by administering a SEMA3A antisense, shRNA or siRNA.
In a further aspect, the present invention concerns a compound for the prevention or treatment of inflammation wherein the compound a) reduces NRP1 expression or activity; or b) reduces NRP1 ligand expression or activity.
In another aspect, the present invention relates to a compound for preventing or reducing hyperactivation of innate immune response, wherein the compound a) reduces NRP1 expression or activity; or b) reduces NRP1 ligand expression or activity.
In an embodiment, the compound is: i) A SEMA3A antibody; ii) A NRP1 antibody; iii) A NRP1 trap; iv) A SEMA3A antisense, shRNA or siRNA; or v) A NRP1 antisense, shRNA or siRNA. In another particular embodiment the compound is a NRP1 antibody or a NRP1 trap and said compound does not substantially reduce the binding of VEGF to NRP1.
In a particular embodiment, the compound is a NRP1 trap. In an embodiment, the NRP1 trap is as set forth in
In another embodiment, the NRP1 trap of the present invention inhibits the binding of SEMA3A to NPR-1 but does not substantially inhibit the binding of VEGF to NRP1. In an embodiment, such NRP1 trap comprises the a1a2 domain of NRP1 but does not comprise the b1 and/or b2 subdomain(s) of NRP1. In another embodiment, such trap comprises a mutation in domain b1 at a position corresponding to tyrosine 297 of the NRP1 amino acid sequence as set forth in
In specific embodiments, the NRP1 trap of the present invention: a) comprises domains a1, a2, b1, b2 and c and of NRP1; b) comprises domains a1, a2, b1 and b2 of NRP1; c) comprises domains a1, a2 and b1 of NRP1; d) comprises domains a1 and a2 of NRP1; e) comprises domain b1, wherein the b1 domain comprises a mutation in amino acid corresponding to tyrosine 297 of NRP1 which reduces or abrogates the binding to VEGF; f) comprises domain b1, wherein the b1 domain comprises a mutation in amino acid corresponding to tyrosine 297 of NRP1 which changes the tyrosine to an alanine; g) does not comprise domain c of NRP1; h) does not comprise domain b1 of NRP1; i) does not comprise domains b1 and b2 of NRP1; or j) does not comprise domains b1, b2 and c of NRP1.
In an embodiment, the NRP1 trap of the present invention comprises: (i) amino acids 1-856 (preferably, 22 to 856) of the NRP1 polypeptide set forth in
In another aspect, the present invention provides compositions for i) treating and preventing inflammation or ii) for preventing or reducing the hyperactivation of the innate immune response, comprising one or more compounds of the present invention together with a pharmaceutical carrier.
The present invention also relates to the use of one or more compounds of the present invention in the manufacture of a medicament for i) treating and preventing inflammation or ii) for preventing or reducing the hyperactivation of the innate immune response.
In a related aspect, the present invention concerns the use of one or more compounds of the present invention for i) treating and preventing inflammation or ii) for preventing or reducing the hyperactivation of the innate immune response.
In a particular embodiment, the methods, compounds (e.g., NRP1 polypeptide traps, nucleic acids encoding same, vectors, cells comprising vectors, etc.), compositions and uses of the present invention are for treating or preventing inflammatory diseases and conditions selected from the group consisting of septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis, diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease or stroke.
In an embodiment, the methods, compounds, compositions and uses of the present invention are for treating or preventing septic shock, cerebral ischemia or stroke.
More specifically, in accordance with the present invention, there is provided the following items:
1. A method of treating or preventing inflammation comprising inhibiting NRP1-dependent cell-signaling in a subject.
2. A method of preventing or reducing hyperactivation of innate immune response comprising inhibiting NRP1-dependent cell-signaling in a subject.
3. The method of item 2, wherein said hyperactivation of innate immune response comprises i) secretion of IL-6, IL-1β and TNFα and/or recruitment of mononuclear phagocytes (MPs).
4. The method of any one of items 1-3, wherein inhibiting NRP1-dependent cell-signaling comprises: (a) reducing NRP1 expression or activity; and/or (b) reducing NRP1 ligand expression or activity; wherein said NRP1 ligand is SEMA3A, VEGF and/or TGF-β.
5. The method of item 4, wherein the method comprises (i) reducing NRP1 activity by inhibiting the binding of NRP1 to at least one NRP1 ligand.
6. The method of item 5, wherein the NRP1 ligand is SEMA3A, VEGF or TGF-β.
7. The method of item 5 or 6, wherein inhibiting the binding of NRP1 to at least one NRP1 ligand comprises administering an anti-NRP1 antibody or an NRP1 trap, wherein said trap comprises a NRP1 polypeptide or a functional fragment or variant thereof.
8. The method of item 7, wherein said NRP1 polypeptide corresponds to soluble NRP1 isoform 2.
9. The method of item 8, wherein said soluble NPR1 isoform 2 comprises or consists essentially of a polypeptide having an amino acid sequence as set forth in
10. The method of item 7, wherein said NRP1 polypeptide corresponds to the extracellular domain of an NRP1 isoform 1 polypeptide.
11. The method of item 10, wherein said NRP1 isoform 1 polypeptide is as set forth in
12. The method of any one of items 7 to 11, wherein said NRP1 trap comprises an NRP1 polypeptide comprising (i) amino acids 22 to 609 of a NRP1 polypeptide as set forth in SEQ ID NO: 65; (ii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 66; (iii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 69 (iv) or a functional fragment or functional variant of (i), (ii) or (iii).
13. The method of any one of items 7 to 12, wherein said anti-NRP1 antibody inhibits the binding of SEMA3A to NPR-1 but does not substantially inhibit the binding of VEGF to NRP1 and wherein said NRP1 trap binds to SEMA3A but does not substantially bind to VEGF165 or has a reduced binding affinity for VEGF165 compared to SEMA3A binding affinity.
14. The method of item 13, wherein said NRP1 trap (i) lacks completely or partially domain b1 and/or b2 of NRP1; or (ii) comprises at least one amino acid point mutation which inhibits VEGF binding to NRP1.
15. The method of item 13, wherein said anti-NRP1 antibody does not bind to domain b1 and/or b2 of NRP1.
16. The method of item 14, wherein said point mutation comprises (a) an amino acid substitution or deletion in domain b1 at an amino acid residue corresponding to tyrosine 297 of an NRP1 amino acid sequence set forth in
17. The method of item 16, wherein said point mutation is a Y297A substitution; a D320K substitution and/or a E319K substitution.
18. The method of any one of item 7 to 12, wherein said NRP1 trap: (a) comprises domains a1, a2, b1, b2, and c and of said NRP1 polypeptide; (b) comprises domains a1, a2, b1 and b2 of said NRP1 polypeptide; (c) comprises domains a1, a2, and b1 of said NRP1 polypeptide; (d) comprises domains a1 and a2 said NRP1 polypeptide; (f) comprises domain b1 of said NRP1 polypeptide, wherein said domain b1 comprises at least one point mutation at an amino acid residue corresponding to (i) tyrosine 297; (ii) aspartic acid 320 and/or (iii) glutamic acid 319, of a NRP1 polypeptide comprising an amino acid sequence as set forth in
19. The method of item 18, wherein (i) said domain a1 comprises or consists essentially of an amino acids sequence corresponding to amino acids 27 to 141 of an NRP1 polypeptide as set forth in
20. The method of item 18, wherein (i) said domain a1 comprises or consists essentially of an amino acids sequence corresponding to amino acids 22 to 148 of an NRP1 polypeptide as set forth in
21. The method of item 7, wherein said method comprises inhibiting the binding of NRP1 to at least one NRP1 ligand by administering a NRP1 trap consisting essentially of a trap as set forth in Table 1 or a functional variant thereof.
22. The method of any one of items 7 to 20, wherein said NRP1 trap further comprises a protein purification domain.
23. The method of 22, wherein said purification domain is a polyhistidine tag.
24. The method of any one of items 7 to 20, wherein said NRP1 trap further comprises a FC domain.
25. The method of any one of items 22 to 24, wherein said NRP1 trap comprises a protease or peptidase cleavage site enabling said protein purification domain or FC domain to be removed from said NRP1 trap.
26. The method of item 25, wherein said protease or peptidase is a TEV protease cleavage site.
27. The method of item 26, wherein said TEV protease cleavage site comprises the amino acid sequence GSKENLYFQG.
28. The method of item 4, wherein the method comprises reducing NRP1 ligand expression or activity, and wherein the NRP1 ligand is SEMA3A.
29. The method of item 28, comprising reducing SEMA3A activity by inhibiting SEMA3A binding to NRP1 by administering an anti-SEMA3A antibody which binds to the SEMA domain of SEMA3A.
30. The method of 4, wherein said method comprises reducing NRP1 expression by administering a NRP1 antisense, shRNA or siRNA.
31. The method of 4, wherein said method comprises reducing SEMA3A expression by administering a SEMA3A antisense, shRNA or siRNA.
32. A NRP1 polypeptide trap comprising s a NRP1 polypeptide or a functional fragment or variant thereof which binds to SEMA3A, VEGF165 and/or TGF-β.
33. The NRP1 polypeptide trap of item 32, wherein said NRP1 polypeptide corresponds to soluble NRP1 isoform 2.
34. The NRP1 polypeptide trap of item 33, wherein said soluble NPR1 isoform 2 comprises or consists essentially of a polypeptide having an amino acid sequence as set forth in
35. The NRP1 polypeptide trap of item 34, wherein said NRP1 polypeptide corresponds to the extracellular domain of an NRP1 isoform 1 polypeptide.
36. The NRP1 polypeptide trap of item 35, wherein said NRP1 isoform 1 polypeptide is as set forth in
37. The NRP1 polypeptide trap of any one of items 32 to 36, wherein said NRP1 trap comprises an NRP1 polypeptide comprising (i) amino acids 22 to 609 of a NRP1 polypeptide as set forth in SEQ ID NO: 65; (ii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 66; (iii) amino acids 22 to 859 of a NRP1 polypeptide as set forth in SEQ ID NO: 69 (iv) or a functional fragment or functional variant of (i), (ii) or (iii).
38. The NRP1 polypeptide trap of item any one of items 32 to 37, wherein NRP1 trap binds to SEMA3A but does not substantially bind to VEGF165 or has a reduced binding affinity for VEGF165 as compared to SEMA3A binding affinity.
39. The NRP1 polypeptide trap of item 38, wherein said NRP1 trap (i) lacks completely or partially domain b1 and/or b2 of NRP1; or (ii) comprises at least one amino acid point mutation which inhibits VEGF binding to NRP1.
40. The NRP1 polypeptide trap of item 39, wherein said point mutation comprises (a) an amino acid substitution or deletion in domain b1 at an amino acid residue corresponding to tyrosine 297 of an NRP1 amino acid sequence set forth in
41. The NRP1 polypeptide trap of item 40, wherein said mutation point is a Y297A substitution; a D320K substitution and/or a E319K substitution.
42. The NRP1 polypeptide trap of any one of items 32 to 38, wherein said trap: (a) comprises domains a1, a2, b1, b2, and c and of said NRP1 polypeptide; (b) comprises domains a1, a2, b1 and b2 of said NRP1 polypeptide; (c) comprises domains a1, a2, and b1 of said NRP1 polypeptide; (d) comprises domains a1 and a2 said NRP1 polypeptide; (e) comprises domain b1 of said NRP1 polypeptide, wherein said domain b1 comprises at least one point mutation at an amino acid residue corresponding to (i) tyrosine 297; (ii) aspartic acid 320 and/or (iii) glutamic acid 319, of a NRP1 polypeptide comprising an amino acid sequence as set forth in
43. The NRP1 polypeptide trap of item 42, wherein (i) said domain a1 comprises or consists essentially of an amino acids sequence corresponding to amino acids 27 to 141 of an NRP1 polypeptide as set forth in
44. The NRP1 polypeptide trap of item 42, wherein (i) said domain a1 comprises or consists essentially of an amino acids sequence corresponding to amino acids 22 to 148 of an NRP1 polypeptide as set forth in
45. The NRP1 polypeptide trap of item 32, wherein said trap consists essentially of a trap as set forth in Table 1 or a functional variant thereof.
46. The NRP1 polypeptide trap of any one of items 32 to 44, wherein said trap further comprises a protein purification domain.
47. The NRP1 polypeptide trap of item 46, wherein said purification domain is a polyhistidine tag.
48. The NRP1 polypeptide trap of any one of items 32 to 47, wherein said NRP1 trap further comprises a FC domain.
49. The NRP1 polypeptide trap of any one of items 46 to 48, wherein said NRP1 trap comprises a protease or peptidase cleavage site enabling said protein purification domain or FC domain to be removed from said NRP1 trap.
50. The NRP1 polypeptide trap of item 49, wherein said protease or peptidase cleavage site is a TEV protease cleavage site.
51. The NRP1 polypeptide trap of item 50, wherein said TEV protease cleavage site comprises the amino acid sequence GSKENLYFQG.
52. A nucleic acid encoding the NRP1 polypeptide trap of any one of items 32-51.
53. An expression vector comprising the nucleic acid of item 52.
54. A host cell comprising the vector of item 53.
55. A composition comprising the NRP1 polypeptide trap of any one of items 32 to 51, the nucleic acid of item 52, the vector of item 53 or the host cell of item 54 and a suitable carrier.
56. The composition of item 55 for (ii) for preventing or treating inflammation, or (ii) preventing or reducing hyperactivation of innate immune response.
57. A compound for preventing or treating inflammation, wherein said compound: (a) reduces NRP1 expression or activity; and/or (b) reduces NRP1 ligand expression or activity.
58. A compound for preventing or reducing hyperactivation of innate immune response, wherein said compound: (a) reduces NRP1 expression or activity; and/or (b) reduces NRP1 ligand expression or activity.
59. The compound of item 57 or 58, wherein said compound is: (i) A anti SEMA3A antibody; (ii) An anti VEGF165 antibody; (iii) A anti NRP1 antibody (iv) A NRP1 trap; (v) A SEMA3A antisense, shRNA or siRNA; (vi) A NRP1 antisense, shRNA or siRNA; or (vii) A VEGF antisense, shRNA or siRNA.
60. The compound item 59, wherein said compound is an NRP1 polypeptide trap.
61. A composition for treating or preventing inflammation comprising a compound as defined in any one of items 57-60 and a suitable carrier.
62. A composition for preventing or reducing hyperactivation of innate immune response comprising a compound as defined in of any one of items 57-60 and a suitable carrier.
63. Use of the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54 the compound of any one of items 57-60 or the composition of any one of items 55, 61 and 62 in the manufacture of a medicament for preventing or treating inflammation.
64. Use of the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54 the compound of any one of items 57-60 or the composition of any one of items 55, 61 and 62 in the manufacture of a medicament for preventing or treating inflammation.
65. Use of the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54 the compound as defined in any one of items 57-60 or the composition as defined in any one of items 55, 61 and 62 for preventing or treating hyperactivation of innate immune response.
66. Use of a the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54 the compound as defined in any one of items 57-60 or the composition as defined in any one of items 55, 61 and 62 for preventing or reducing hyperactivation of innate immune response.
67. The method of any one of items 1-31, wherein said subject suffers or is likely to suffer from septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis, diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease or stroke.
68. The method of any one of items 1-31, the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54, a compound as defined in any one of items 57-60 or a composition as defined in any one of items 55, 61 and 62 wherein said method, NRP1 polypeptide trap, nucleic acid, vector, host cell, compound, composition or use is for treating or preventing septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis, diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease or stroke.
69. The method of any one of items 1-31, the NRP1 polypeptide trap of any one of items 32-51, the nucleic acid of item 52, the vector of item 53, the host cell of item 54, a compound as defined in any one of items 57-60 or a composition as defined in any one of items 55, 61 and 62 wherein said method, NRP1 polypeptide trap, nucleic acid, vector, host cell, compound, composition or use is for treating or preventing septic shock or stroke.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
The present inventors have identified a subset of mononuclear phagocytes (MPs) that responds to local chemotactic cues such as SEMA3A that are conserved between central neurons, vessels and immune cells. NRP1 expressing MP's were shown to enter the site of injury and to contribute to (i) tissue damage and/or (ii) pathological activation of the innate immune response in models of inflammatory conditions including various forms of inflammatory, proliferative retinopathies (e.g., proliferative diabetic retinopathy, retinopathy of prematurity and age-related macular degeneration), septic shock and cerebral ischemia/stroke.
The inventors demonstrated that stressed retinal neurons and neural tissue have the inherent ability to modulate the local innate immune response via unconventional chemotactic agents. NRP1 on microglia was found to be a potent chemoattractive receptor for SEMA3A, and VEGF and inhibition of NRP1 signaling in innate immune cells (e.g., using NRP1-derived traps or NRP1 or SEMA3A antibodies) resulted in protection against MP's induced inflammation and tissue damage.
Patients suffering from late stage proliferative diabetic retinopathy (PDR) were shown to produce elevated levels of SEMA3A which counterintuitively acts as a potent attractant for Neuropilin-1 (NRP1)-positive MPs. These pro-angiogenic MPs are selectively recruited to sites of pathological neovascularization in response to locally produced SEMA3A as well as VEGF and TGF-β. Furthermore, SEMA3A was shown to be up-regulated in several organs during septic shock and to induce secretion of inflammatory cytokines by MP's. Inhibition of NRP1 also reduced the production of proinflammatory cytokines in sepsis.
Finally NRP1-positive MPs were shown to play a critical role in inflammatory disease progression. Inhibition/abrogation of NRP1 myeloid-dependent activity was shown to protect against neovascular retinal disease (vascular degeneration and pathological neovascularization), septic shock and neural damages secondary to cerebral ischemia/stroke.
Together, these findings underscore the role of NRP1-positive MPs and their ligands in inflammation (and in particular in neuroinflammation) and demonstrate the therapeutic benefit of inhibiting NRP1 cell signaling to limit hyperactivation of innate immune response (e.g., tissue damage at the site of injury through recruitment of microglia/macrophages and/or induction of production and/or secretion of proinflammatory cytokines, and/or vascular leakage/edema). The present findings finds applications in the prevention and treatment of diseases and conditions characterized by sustained (e.g., chronic, persistent) or excessive/pathological inflammation involving MP recruitment and proinflammatory cytokines production and secretion such as septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis and neuroinflammatory conditions such as diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease and stroke.
Inhibition of NRP1-Mediated Cellular Activity
The present inventors have found that by inhibiting NRP1-dependent cell signaling (and in particular SEMA3A-mediated cell signaling), it is possible to protect against (prevent or treat) inflammatory diseases and conditions such as those involving hyperactivation of the innate immune response. In particular, inhibition of NRP1-mediated cell-signaling reduces the unwanted (pathological) recruitment of mononuclear phagocytes (MPs, e.g., microglia, macrophages) and the production/secretion of proinflammatory cytokines which contribute to tissue damage (e.g., increased vascular degeneration, pathologic neovascularization, cell death or cell damages), inflammation and edema.
Thus, in an aspect, the present invention relates to a method of treating or preventing inflammation comprising inhibiting NRP1-dependent cell-signaling. In a particular aspect, the inflammation is neuroinflammation.
As used herein, the term “inflammation” means a disease or condition which involves the activation of the innate immune response comprising i) the recruitment of mononuclear phagocytes (e.g., microglia or macrophages) expressing the NRP1 receptor at the site of inflammation or injury; and/or ii) the NRP1 dependent production/secretion of pro-inflammatory cytokines (e.g., IL-1β, TNF-α, IL-6). The classical signs of acute inflammation are pain, heat, redness, swelling, and loss of function. Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged (sustained) inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. Non-limiting examples of inflammatory conditions which may be treated or prevented in accordance with methods of the present invention include septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis and neuroinflammatory conditions such as diabetic retinopathy (including proliferative diabetic retinopathy (PDR)), age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease and stroke.
In a particular embodiment the inflammatory disease or condition is not a retinopathy. In another embodiment, the inflammatory disease or condition is not diabetic retinopathy. In another embodiment, the inflammatory disease or condition is not macular edema. In another embodiment, the inflammatory disease or condition is not diabetic macular edema.
In a related aspect, the present invention concerns a method of inhibiting hyperactivation (or pathological activation) of the innate immune response comprising inhibiting NRP1-dependent cell-signaling. Such an hyperactivation of innate immune response, is typically associated with acute or chronic activation of any given cell population of the immune system (innate and adaptive, e.g., mononuclear cell recruitment in the organ/tissue) beyond levels required to maintain tissue homeostasis. This is often accompanied by heightened production of cytokines (e.g., TNF-alpha, IL-6), increased vascular permeability, and may result in compromised tissue function.
In another aspect, the present invention concerns a method of treating or preventing vascular degeneration comprising inhibiting NRP1-dependent cell-signaling.
In a further aspect, the present invention concerns a method of treating or preventing pathological neovascularization comprising inhibiting NRP1-dependent cell-signaling.
In another aspect, the present invention concerns a method of treating or preventing septic shock comprising inhibiting NRP1-dependent cell-signaling.
In a yet another aspect, the present invention concerns a method of treating or preventing neural damages secondary to cerebral ischemia/stroke comprising inhibiting NRP1-dependent cell-signaling.
Because NRP1-mediated cell signaling (e.g., MPs recruitment and production/secretion of pro-inflammatory cytokines) depends on the binding of NRP1 to its ligands (e.g., SEMA3A, VEGF and/or TGF-β), inhibition of NRP1-mediated cellular signaling can be achieved in at least two ways: i) by targeting the expression or activity of NRP1 directly (through the use of NRP1 antibodies, NRP1 derived traps or the like); or ii) by targeting the expression or activity of one or more of its ligands (e.g., SEMA3A, VEGF and/or TGF-β).
In embodiments, the above methods comprise preferentially or specifically inhibiting SEMA3A-mediated cell signalling. “Preferentially inhibiting” means that the level of inhibition of SEMA3A-mediated cell signalling is greater than that of other NRP1 ligands (e.g., VEGF165 and TGF-beta). In certain aspects, methods of the present invention substantially do not reduce or inhibit VEGF (e.g., VEGF165) and/or TGF-beta-mediated cell signalling that occur through the interaction with NRP1. In embodiments, compounds of the present invention (e.g., NRP1 traps) “preferentially bind” to one ligand over the others (e.g., preferentially bind SEMA3A over VEGF). Such preferential interaction may be determined by measuring the dissociation constant (Kd) for each ligand. In embodiments, interaction for one ligand (e.g., SEMA3A) over the others (e.g., VEGF) is at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 28, 20, 22, 25, 30, 35, 40, 45, 50, 60, 75, 80, 100, 200, 300, 400, 500, 1000 times greater or more. In embodiments the kD (e.g., in nM) for one ligand is at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 28, 20, 22, 25, 30, 35, 40, 45, 50, 60, 75, 80, 100, 200, 300, 400, 500, 1000 times smaller than the kD for one or more of the other ligands (e.g., VEGF).
In an embodiment, methods of the present invention comprise administration to a subject likely to suffer from inflammation (e.g., likely to suffer from an inflammatory disease or condition). In other embodiment, methods of the present invention comprise administration to a subject diagnosed from inflammation (e.g., likely to suffer from an inflammatory disease or condition). In an embodiment, the subject is a mammal, preferably a human.
NRP1 Traps
Inhibition of NRP1-mediated cellular signaling can be achieved using NRP1 traps of the present invention. As used herein, the terms, “NRP1 trap”, or “NRP1 polypeptide trap” encompass naturally occurring soluble NRP1 polypeptide (e.g., such as NRP1 secreted isoform b
NRP1 traps of the present invention may or may not comprise further polypeptide domains (e.g., purification domains). Exemplary traps lacking purification domains and comprising only NRP1-derived sequences are shown in
Given that NRP1 distinctly regulates the effects of its ligands on signal transduction and cellular responses, it may be advantageous to specifically inhibit the binding of one specific ligand to NRP1 but not that of the others. For example, as shown herein, at early time points of retinal disease, where SEMA3A levels are elevated, VEGF levels remain low and relatively unchanged compared to non-diabetic controls. Also, in septic shock, SEMA3A was the sole NRP1 ligand which had a long term effect and stayed up-regulated for more than 24 hours following induction of sepsis. Thus, given the differences in expression kinetics for each ligand and the fact that neutralization of one ligand (e.g., VEGF) may be ineffective in certain conditions (or be associated with undesired side effects), specific inhibition of one ligand (e.g., SEMA3A) binding to NRP1, (but not that of the other(s) (e.g., VEGF)) is advantageous. Thus, in certain aspects of the methods of the present invention, inhibition of SEMA3A-mediated cell signaling, is accomplished by providing NRP1 Traps having greater affinity for SEMA3 than VEGF or to which VEGF (e.g., VEGF165) does not bind or does not bind substantially.
Accordingly, in an embodiment, the soluble NRP1 polypeptide or functional fragment or variant thereof (NRP1 trap) of the present invention binds to all natural ligands of NRP1 (e.g., SEMA3A, VEGF and TGF-beta, e.g., a soluble NRP1 trap comprising the extracellular domain (e.g., amino acids 22-856 or 22-959 of SEQ ID NO: 66 or 69), Trap 1, (SEQ ID NO: 1) or Trap G (SEQ ID NO: 38)—See also,
In another embodiment, the NRP1 trap of the present invention is a polypeptide which binds to SEMA3A but not to VEGF. For example the NRP1 trap may comprise the a1 (e.g., SEQ ID NO:71) and/or a2 subdomain(s) (e.g., SEQ ID NO:72) which bind(s) to SEMA3A but not the b1 (e.g., SEQ ID NO:73) and/or b2 (e.g., SEQ ID NO: 74) subdomain(s) required for VEGF binding (e.g., Trap M, (SEQ ID NO: 42), Trap N (SEQ ID NO: 44), Trap 12/Trap K (SEQ ID NO: 23), Trap 4 (SEQ ID NO:7), Trap 7/C (SEQ ID NO: 13), See also,
In another embodiment, the NRP1 trap is a soluble NRP1 polypeptide or functional fragment or variant thereof which binds to VEGF but not to SEMA3A. For example, the NRP1 trap may comprise the b1 (e.g., SEQ ID NO: 73) and/or b2 (e.g., SEQ ID NO: 74) domain(s) to bind to VEGF but not the a1 (e.g., SEQ ID NO: 71) and/or a2 (e.g., SEQ ID NO: 72) subdomain(s) which bind to SEMA3A. In an embodiment, the NRP1 trap comprises domains b1b2 corresponding to amino acids 276 to 589 of the NRP1 amino acid sequence set forth in
In an embodiment, the soluble NRP1 polypeptide or functional fragment thereof comprises or consists of traps as set forth in
In preferred embodiments, the NRP1 traps of the present invention lack the transmembrane domain (e.g., corresponding to amino acids residues 860 to 883 of the NRP1 polypeptide sequences shown in
The soluble NRP1 polypeptide or functional fragment or variant thereof of the present invention may comprise one or more additional polypeptide domain(s) to increase in vivo stability and/or facilitate purification. For example, NRP1 traps of the present invention may include a FC domain (or part thereof such as the human FC domain set forth in SEQ ID NO: 37.) or a purification tag (e.g., a 6×-histidine tag). Such additional polypeptide domain(s) may be linked directly or indirectly (through a linker) to the soluble NRP1 polypeptide or functional fragment thereof.
The soluble NPR1 polypeptide or functional fragment thereof of the present invention may optionally include one or more polypeptide linkers. Such linkers may be used to link one or more additional polypeptide domain(s) to the soluble polypeptide of the present invention (e.g., a polypeptide domain which increases the stability of the polypeptide in vivo and/or a domain which facilitates purification of the polypeptide). Linker sequence may optionally include peptidase or protease cleavage sites which may be used to remove one or more polypeptide fragments or domains (e.g., removal of purification tag prior to in vivo administration of the soluble NRP1 polypeptides or functional fragment thereof). One non-limiting example of a linker or domain which enables cleavage of the polypeptide and removal of, for example, polypeptide domain(s) (e.g., 6× his tag purification domain) includes a polypeptide comprising a TEV protease cleavage site (e.g., GSKENLYFQ'G, SEQ ID NO:76). Many other TEV cleavage sites are known and many other protease/peptidase cleavage sites are known to the skilled person and may be introduced in the polypeptides of the present invention to optionally remove one or more polypeptide domains or fragments.
Polypeptide linkers may also be used to replace totally or partially domains which are normally found in the wild-type NRP1 polypeptide but which are absent in the soluble NRP1 polypeptide or functional fragment thereof of the present invention. For example, in the NRP1 traps of the present invention, synthetic linkers may replace totally or partially domains a1, a2, b1, b2 and c. The length of the linker may correspond to the entire length of the domain removed or to a portion of it. Such linkers may increase protein folding, stability or binding to NRP1 ligands. Non-limiting examples of NRP1 traps comprising linkers are shown in
In an embodiment, the NRP1 trap of the present invention comprises: (i) amino acids 1-856 (preferably, 22 to 856) of the NRP1 polypeptide set forth in
In a related aspect, the present invention provides nucleic acids encoding the NRP1 traps (e.g., traps listed in Table 1 and shown on
As used herein, a “functional fragment” or “functional variant” of NRP1 (e.g., a functional fragment of soluble NRP1 polypeptide or polynucleotide of the present invention such as an NRP1) refers to a molecule which retains substantially the same desired activity as the original molecule but which differs by any modifications, and/or amino acid/nucleotide substitutions, deletions or additions (e.g., fusion with another polypeptide). Modifications can occur anywhere including the polypeptide/polynucleotide backbone (e.g., the amino acid sequence, the amino acid side chains and the amino or carboxy termini). Such substitutions, deletions or additions may involve one or more amino acids or in the case of polynucleotide, one or more nucleotide. The substitutions are preferably conservative, i.e., an amino acid is replaced by another amino acid having similar physico-chemical properties (size, hydrophobicity, charge/polarity, etc.) as well known by those of ordinary skill in the art. Functional fragments of the soluble NRP1 include a fragment or a portion of a soluble NRP1 polypeptide (e.g., the a1 and/or a2 domain(s)) or a fragment or a portion of a homologue or allelic variant of NRP1 which retains inhibiting activity, i.e., binds to SEMA3A, VEGF and/or TGF-β and inhibits the transduction of NRP1-mediated cellular activity. Non-limiting examples of NRP1-mediated cellular activity include i) vascular hyperpermeability; ii) MPs activation and recruitment; iii) inducement of apoptosis; iv) induction of pro-inflammatory cytokines (e.g., TNF-α, IL-1β) production and/or secretion. In an embodiment, the NRP1 polypeptide is at least 80, 85, 88, 90, 95, 98 or 99% identical to the polypeptide sequence of
Antibodies
NRP1 cellular activity can be inhibited by using an agent which blocks NRP1 binding to one or more of its ligands (e.g., SEMA3A, VEGF and/or TGF-β). One example of such agent is an antibody which binds to NRP1 and blocks the binding of NRP1 to SEMA3A, VEGF and/or TGF-β.
Alternatively, inhibition of NRP1-mediated cellular signaling can be achieved by using an agent which blocks the binding of an NRP1 ligand to the NRP1 polypeptide. Non-limiting examples of such agent includes an antibody which binds to SEMA3A, VEGF or TGF-β and blocks their respective binding to NRP1.
In a particular aspect of the present invention, antibodies targeting NRP1 block SEMA3A binding to the receptor but do not substantially interfere with VEGF and/or TGF-β binding to NRP1. In an embodiment, the anti NRP1 antibody binds to the a1a2 domains of the NRP1 polypeptide. In another embodiment, the anti NRP1 antibody binds to subdomains a1 or a2 of the NRP1 polypeptide.
As noted above, anti SEMA3A antibodies may be used to inhibit (i.e., reduce completely or partially) NRP1-mediated cellular signaling by blocking SEMA3A binding to NRP1. Useful anti SEMA3A antibodies bind to the SEMA domain of SEMA3A and block the interaction with NRP1. In embodiments the anti-SEMA3A antibodies used in accordance with the present invention include antibodies binding to SEMA3A polypeptide domains comprising amino acid residues 252-260, 359-366 or 363-380 of SEMA3A. SEMA3A antibodies which inhibit the binding of SEMA3A to NRP1 are known in art and may be used in accordance with the present invention.
As used herein, the expression “anti NRP1 antibody” refers to an antibody that specifically binds to (interacts with) a NRP1 protein and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as the NRP1 protein. Similarly, the expression “anti SEMA3A antibody”, “anti VEGF antibody” or “anti TGF-β antibody” refers to an antibody that specifically binds to (interacts with) a SEMA3A, VEGF or TGF-β protein respectively and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as the targeted SEMA3ANEGF/TGF-β protein.
Antibodies that can be used in accordance with the present invention include polyclonal, monoclonal, humanized as well as chimeric antibodies. The term antibody or immunoglobulin is used in the broadest sense, and covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies and antibody fragments so long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), nanobodies, shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies™, antibody-T-cell epitope fusions (Troybodies) or Peptibodies.
Anti-human NRP1/sem3A/VEGF/TGF-β antibodies have been previously prepared and are also commercially available from various sources including Santa Cruz, AbCam, and Cell Signaling.
In general, techniques for preparing antibodies (including monoclonal antibodies, hybridomas and humanized antibodies when their sequences are known) and for detecting antigens using antibodies are well known in the art and various protocols are well known and available.
Inhibition of the Expression of NRP1 or NRP1 Ligands
Various approaches are available for decreasing the expression (at the mRNA or protein level) of NRP1 or its ligands (e.g., SEMA3A, VEGF or TGF-β) to inhibit NRP1 mediated cell signaling and thus reduce inflammation and hyperactivation of innate immune response (i.e., i) production and/or secretion of pro-inflammatory cytokines; ii) recruitment of mononuclear phagocytes (MPs); iii) vascular hyperpermeabilization; and/or iv) edema, v) neuronal damage, choroidal neovascularization etc.). Non-limiting example includes the use of small hairpin shRNA (RNAi), antisense, ribozymes, TAL effectors targeting the NRP1, SEMA3A, VEGF or Tgf-β promoter or the like.
Expression in cells of shRNAs, siRNAs, antisense oligonucleotides or the like can be obtained by delivery of plasmids or through viral (e.g., lentiviral vector) or bacterial vectors.
Therefore, in alternative embodiments, the present invention provides antisense, shRNA molecules and ribozymes for exogenous administration to effect the degradation and/or inhibition of the translation of mRNA of interest. Preferably, the antisense, shRNA molecules and ribozymes target human NRP1, SEMA3A, VEGF and/or Tgf-β expression. Examples of therapeutic antisense oligonucleotide applications include: U.S. Pat. No. 5,135,917, issued Aug. 4, 1992; U.S. Pat. No. 5,098,890, issued Mar. 24, 1992; U.S. Pat. No. 5,087,617, issued Feb. 11, 1992; U.S. Pat. No. 5,166,195 issued Nov. 24, 1992; U.S. Pat. No. 5,004,810, issued Apr. 2, 1991; U.S. Pat. No. 5,194,428, issued Mar. 16, 1993; U.S. Pat. No. 4,806,463, issued Feb. 21, 1989; U.S. Pat. No. 5,286,717 issued Feb. 15, 1994; U.S. Pat. Nos. 5,276,019 and 5,264,423; BioWorld Today, Apr. 29, 1994, p. 3.
Preferably, in antisense molecules, there is a sufficient degree of complementarity to the mRNA of interest to avoid non-specific binding of the antisense molecule to non-target sequences under conditions in which specific binding is desired, such as under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted. The target mRNA for antisense binding may include not only the information to encode a protein, but also associated ribonucleotides, which for example form the 5′-untranslated region, the 3′-untranslated region, the 5′ cap region and intron/exon junction ribonucleotides. A method of screening for antisense and ribozyme nucleic acids that may be used to provide such molecules as Shc inhibitors of the invention is disclosed in U.S. Pat. No. 5,932,435.
Antisense molecules (oligonucleotides) of the invention may include those which contain intersugar backbone linkages such as phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages, phosphorothioates and those with CH2—NH—O—CH2, CH2—N(CH3)—O—CH2 (known as methylene(methylimino) or MMI backbone), CH2—O—N(CH3)—CH2, CH2—N(CH3)—N(CH3)—CH2 and O—N(CH3)—CH2—CH2 backbones (where phosphodiester is O—P—O—CH2). Oligonucleotides having morpholino backbone structures may also be used (U.S. Pat. No. 5,034,506). In alternative embodiments, antisense oligonucleotides may have a peptide nucleic acid (PNA, sometimes referred to as “protein nucleic acid”) backbone, in which the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone wherein nucleosidic bases are bound directly or indirectly to aza nitrogen atoms or methylene groups in the polyamide backbone (Nielsen et al., 1991, Science 254:1497 and U.S. Pat. No. 5,539,082). The phosphodiester bonds may be substituted with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.
Oligonucleotides may also include species which include at least one modified nucleotide base. Thus, purines and pyrimidines other than those normally found in nature may be used. Similarly, modifications on the pentofuranosyl portion of the nucleotide subunits may also be effected. Examples of such modifications are 2′-O-alkyl- and 2′-halogen-substituted nucleotides. Some specific examples of modifications at the 2′ position of sugar moieties which are useful in the present invention are OH, SH, SCH3, F, OCN, O(CH2)nNH2 or O(CH2)nCH3 where n is from 1 to about 10; C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. One or more pentofuranosyl groups may be replaced by another sugar, by a sugar mimic such as cyclobutyl or by another moiety which takes the place of the sugar.
In some embodiments, the antisense oligonucleotides in accordance with this invention may comprise from about 5 to about 100 nucleotide units. As will be appreciated, a nucleotide unit is a base-sugar combination (or a combination of analogous structures) suitably bound to an adjacent nucleotide unit through phosphodiester or other bonds forming a backbone structure.
In a further embodiment, expression of a nucleic acid encoding a polypeptide of interest (e.g., SEMA3A or NRP1), or a fragment thereof, may be inhibited or prevented using RNA interference (RNAi) technology, a type of post-transcriptional gene silencing. RNAi may be used to create a pseudo “knockout”, i.e. a system in which the expression of the product encoded by a gene or coding region of interest is reduced, resulting in an overall reduction of the activity of the encoded product in a system. As such, RNAi may be performed to target a nucleic acid of interest or fragment or variant thereof, to in turn reduce its expression and the level of activity of the product which it encodes. Such a system may be used for functional studies of the product, as well as to treat disorders related to the activity of such a product. RNAi is described in for example published US patent applications 20020173478 (Gewirtz; published Nov. 21, 2002) and 20020132788 (Lewis et al.; published Nov. 7, 2002). Reagents and kits for performing RNAi are available commercially from for example Ambion Inc. (Austin, Tex., USA) and New England Biolabs Inc. (Beverly, Mass., USA).
The initial agent for RNAi in some systems is a dsRNA molecule corresponding to a target nucleic acid. The dsRNA (e.g., shRNA) is then thought to be cleaved into short interfering RNAs (siRNAs) which are 21-23 nucleotides in length (19-21 bp duplexes, each with 2 nucleotide 3′ overhangs). The enzyme thought to effect this first cleavage step has been referred to as “Dicer” and is categorized as a member of the RNase III family of dsRNA-specific ribonucleases. Alternatively, RNAi may be effected via directly introducing into the cell, or generating within the cell by introducing into the cell a suitable precursor (e.g. vector encoding precursor(s), etc.) of such an siRNA or siRNA-like molecule. An siRNA may then associate with other intracellular components to form an RNA-induced silencing complex (RISC). The RISC thus formed may subsequently target a transcript of interest via base-pairing interactions between its siRNA component and the target transcript by virtue of homology, resulting in the cleavage of the target transcript approximately 12 nucleotides from the 3′ end of the siRNA. Thus the target mRNA is cleaved and the level of protein product it encodes is reduced.
RNAi may be effected by the introduction of suitable in vitro synthesized siRNA (shRNAs) or siRNA-like molecules into cells. RNAi may for example be performed using chemically-synthesized RNA. Alternatively, suitable expression vectors may be used to transcribe such RNA either in vitro or in vivo. In vitro transcription of sense and antisense strands (encoded by sequences present on the same vector or on separate vectors) may be effected using for example T7 RNA polymerase, in which case the vector may comprise a suitable coding sequence operably-linked to a T7 promoter. The in vitro-transcribed RNA may in embodiments be processed (e.g. using E. coli RNase III) in vitro to a size conducive to RNAi. The sense and antisense transcripts are combined to form an RNA duplex which is introduced into a target cell of interest. Other vectors may be used, which express small hairpin RNAs (shRNAs) which can be processed into siRNA-like molecules. Various vector-based methods and various methods for introducing such vectors into cells, either in vitro or in vivo (e.g. gene therapy) are known in the art.
Accordingly, in an embodiment expression of a nucleic acid encoding a polypeptide of interest (or a fragment thereof e.g., soluble NRP1, NRP1 derived traps, may be inhibited by introducing into or generating within a cell an siRNA or siRNA-like molecule corresponding to a nucleic acid encoding a polypeptide of interest (e.g. SEMA3A or NRP1), or a fragment thereof, or to an nucleic acid homologous thereto. “siRNA-like molecule” refers to a nucleic acid molecule similar to an siRNA (e.g. in size and structure) and capable of eliciting siRNA activity, i.e. to effect the RNAi-mediated inhibition of expression. In various embodiments such a method may entail the direct administration of the siRNA or siRNA-like molecule into a cell, or use of the vector-based methods described above. In an embodiment, the siRNA or siRNA-like molecule is less than about 30 nucleotides in length. In a further embodiment, the siRNA or siRNA-like molecule is about 21-23 nucleotides in length. In an embodiment, siRNA or siRNA-like molecule comprises a 19-21 bp duplex portion, each strand having a 2 nucleotide 3′ overhang. In embodiments, the siRNA or siRNA-like molecule is substantially identical to a nucleic acid encoding a polypeptide of interest, or a fragment or variant (or a fragment of a variant) thereof. Such a variant is capable of encoding a protein having activity similar to the polypeptide of interest.
A variety of viral vectors can be used to obtain shRNA/RNAi expression in cells including adeno-associated viruses (AAVs), adenoviruses, and lentiviruses. With adeno-associated viruses and adenoviruses, the genomes remain episomal. This is advantageous as insertional mutagenesis is avoided. It is disadvantageous in that the progeny of the cell will lose the virus quickly through cell division unless the cell divides very slowly. AAVs differ from adenoviruses in that the viral genes have been removed and they have diminished packing capacity. Lentiviruses integrate into sections of transcriptionally active chromatin and are thus passed on to progeny cells. With this approach there is increased risk of insertional mutagenesis; however, the risk can be reduced by using an integrase-deficient lentivirus.
Pharmaceutical Compositions and Kits
Agents which inhibit NRP1-dependent cell signaling (i.e., NRP1 inhibitors) of the present invention can be administered to a human subject by themselves or in pharmaceutical compositions where they are mixed with suitable carriers or excipient(s) at doses to treat or prevent the targeted disease or condition or to raise the desired cellular response.
Mixtures of these compounds (e.g., NRP1 trap, antibodies, dominant negative, small inhibitory peptides or the like) can also be administered to the subject as a simple mixture or in suitable formulated pharmaceutical compositions. A therapeutically effective dose further refers to that amount of the compound or compounds sufficient to result in the prevention or treatment of the targeted inflammatory disease or condition (e.g., such as septic shock, arthritis, inflammatory bowel disease (IBD), cutaneous skin inflammation, diabetes, uveitis and neuroinflammatory conditions such as diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity, multiple sclerosis, amyotrophic lateral sclerosis (ALS), age-related cognitive decline/Alzheimer's disease) or to provide the desired cellular or physiological response (e.g., amount sufficient to i) reduce edema, ii) reduce activation/recruitment of mononuclear phagocytes (e.g., microglia or macrophages), iii) reduce production or secretion of inflammatory cytokines (e.g., IL-1β, TNF-α, IL-6, etc.); iv) reduce pathological neovascularization; v) reduce vascular degeneration, etc.).
As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, physiological media, and the like that are physiologically compatible. In embodiments the carrier is suitable for ocular administration. In other embodiments the carrier is suitable for systemic administration. In other embodiments the carrier is suitable for oral administration.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents, such as for ocular, systemic or oral application, is well known in the art. Except insofar as any conventional media or agent is incompatible with the compounds of the invention, use thereof in the compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.
The present invention also concerns kits or commercial packages for use in the methods of the present invention. Such kits may comprises compounds of the present invention (e.g., compounds which inhibit NRP1 cell signaling, including SEMA3A-mediated cell signaling such as traps, antibodies, shRNA cells, vectors, nucleic acids) optionally with instructions to use the kit.
Routes of Administration/Formulations
Suitable routes of administration may, for example, include systemic, oral and ocular (eye drops or intraocular injections). Preferred routes of administration comprise eye drops and intraocular injections for eye conditions, oral for chronic inflammatory conditions and systemic for sepsis and certain neuronal conditions such as stroke. The formulations may also be in the form of sustained release formulations.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
The compounds may be formulated for ocular administration e.g., eye drops or ocular injections. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a cell-specific antibody or other delivery system (e.g., to target for example a specific tissue (e.g., brain) or cell type (e.g., microglia or macrophages)). Nanosystems and emulsions are additional well known examples of delivery vehicles or carriers for drugs. Another example is the Encapsulated Cell Therapy (ECT) delivery system from Neurotech's, for eye diseases. ECT is a genetically engineered ocular implant that enables continuous production of therapeutic proteins to the eye for over 2 years. Additionally, the therapy is reversible by simply removing the implant. The ECT implant is inserted into the vitreous through a single incision and sutured in place in a 20-minute outpatient surgical procedure.
Effective Dosage
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art.
The effective dose of the compound inhibits the cellular signaling function of NRP1 sufficiently to reduce or prevent one or more physiological or cellular responses (e.g., vascular hyperpermeability, blood retinal barrier leakage, edema, MPs activation and/or recruitment, proinflammatory cytokines production and/or secretion, neovascularization, neuronal damage, etc.) or to prevent or treat a given inflammatory disease or condition, without causing significant adverse effects. Certain compounds which have such activity can be identified by in vitro assays that determine the dose-dependent inhibition of NRP1-mediated cell signaling inhibitors (e.g., agents which directly target the expression or activity of NRP1 or agents which targets the expression or activity (e.g., binding) of ligands of NRP1.
For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cellular assays. For example, a dose can be formulated in cellular and animal models to achieve a circulating concentration range that includes the IC50 as determined in cellular assays (i a, the concentration of the test compound which achieves a half-maximal inhibition of the cellular signaling function of NRP1, usually in response to inflammatory mediators such as Il-1β or other activating stimulus such as hypoxia, ischemia, cellular stress, ER stress, etc.
A therapeutically effective amount refers to that amount of the compound that results in amelioration of symptoms in a subject. Similarly, a prophylacticaiiy effective amount refers to the amount necessary to prevent or delay symptoms in a patient (e.g., NRP1-mediated vascular hyperpermeability, spotted and/or blurry vision, pericytes loss, macular edema, retinal swelling, blood retinal barrier leakage, mononuclear phagocytes recruitment, production and secretion of pro-inflammatory cytokines, vascular degeneration, pathological neovascularization, neuronal damage, etc.). Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining the maximum tolerated dose (MTD) and the ED (effective dose for 50% maximal response). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between MID and ED50. Compounds which exhibit high therapeutic indices are preferred. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
Dosage amount and interval may be adjusted individually to provide levels of the active compound which are sufficient to maintain the NRP1 modulating effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; e. g. the concentration necessary to achieve substantial inhibition of SEMA3A expression or activity (e.g., binding to NRP1 receptor) Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
Definitions
For clarity, definitions of the following terms in the context of the present invention are provided.
As used herein, the term “Neuropilin-1 receptor” or “NRP1” receptor refers to neuropilin-1 and its isoforms, and allelic/polymorphic forms (e.g., HGNC: 8004; Entrez Gene: 8829; Ensembl: ENSG00000099250; OMIM: 602069; and UniProtKB: 014786; GenBank Acc. No. AAH07737.1,
The basic structure of neuropilin-1 comprises 5 domains: Three extracellular domains (a1a2 (CUB), b1b2 (FV/FVIII) and c (MAM)), a transmembrane domain and a cytoplasmic domain (See
“Homology” and “homologous” refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid/polynucleotide sequence is “homologous” to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term ‘homologous’ does not infer evolutionary relatedness). Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical. As used herein, a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than about 25% identity, with any of the nucleic acids and polypeptides disclosed herein.
Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information. The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridisation to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. For example, in an embodiment, the compound of the present invention is an antisense/RNAi or shRNA that hybridizes to an NRP1 or SEMA3A nucleic acid sequence (preferably a human sequence).
As used herein the term “treating” or “treatment” in reference to inflammatory diseases or conditions (e.g., retinopathies, cerebral ischemia, stroke, sepsis, ect.) is meant to refer to a reduction/improvement in one or more symptoms or pathological physiological responses associated with said disease or condition. Non-limiting examples include edema, swelling, itching, pain, vascular hyperpermeability; blood retinal barrier integrity, increase in SEMA3A, VEGF and/or TGF-beta expression, mononuclear phagocyte recruitment/chemotaxis, production and/or secretion of proinflammatory cytokines, vascular or neuronal degeneration, etc.
As used herein the term “preventing” or “prevention” in reference to inflammatory diseases or conditions is meant to refer to a reduction in the progression or a delayed onset of at least one symptom associated with the disease or condition.
The articles “a,” “an” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
The term “including” and “comprising” are used herein to mean, and re used interchangeably with, the phrases “including but not limited to” and “comprising but not limited to”.
The terms “such as” are used herein to mean, and is used interchangeably with, the phrase “such as but not limited to”.
The present invention is illustrated in further details by the following non-limiting examples.
Example 1 Materials and Methods (Examples 2-9 and 12)Generation of LyzM-cre/Nrpfl/fl mice. C57131/6 wild-type (WT) were purchased from The Jackson Laboratory. LyzM-Cre (Lyz2tm1(cre)Ifo/J; no. 004781) and NRP1 floxed mice (Nrp1tm2Ddg/J; no. 005247) were purchased from The Jackson Laboratory and bread to obtain LyzM-cre/Nrpfl/fl with NRP1-deficient myeloid cells.
O2-induced retinopathy. Mouse pups (WT or LyzM-Cre (Jackson Laboratory) or LysM-Cre/Nrp1fl/fl) and their fostering mothers (CD1, Charles River) were exposed to 75% O2 from postnatal day 7 (P7) to day 12 and returned to room air (52). This model serves as a proxy to human ocular neovascular diseases such as diabetic retinopathy characterized by a late phase of destructive pathological angiogenesis (53, 54). Upon return to room air, hypoxia-driven neovascularization (NV) develops from P14 onwards (26). Eyes were enucleated at different time points and the retinas dissected for FACS analysis or mRNA analysis as described. In other experiments, dissected retinas were flatmounted and incubated overnight with fluoresceinated isolectin B4 (1:100) in 1 mM CaCl2 to determine extent of avascular area or neovascularization area at P17 using ImageJ and the SWIFT-NV method (55).
FACS of digested retinas and spleen. Retinas from WT or LysM-Cre/Nrp1fl/fl mice were homogenized and incubated in a solution of 750 U/mL DNaseI (Sigma) and 0.5 mg/mL of collagenase D (Roche) for 15 min at 37° C. with gentle shaking. Homogenates were then filtered with a 70 μm cell strainer and washed in PBS+3% fetal bovine serum. Spleen samples were homogenized and incubated with 1 mg/mL of collagenase D for 10 min at 37° C. Homogenates were washed in PBS+3% fetal bovine serum and the pellets were resuspended and incubated in lysis buffer (10 mM KCHO3; 150 mM NH4Cl; 0.1 mM EDTA) for 5 min at room temperature. Cell suspensions (retina or spleen) were incubated with LEAF™ purified anti-mouse CD16/32 (Biolegend) for 15 min at room temperature to block Fc receptors. Cells were then incubated for 30 min at room temperature with the following antibodies: FITC anti-mouse/human CD11b (Biolegend), PE/CY7 anti-mouse Ly-6G/Ly-6C (Gr-1; Biolegend), Pacific Blue™ anti-mouse F4/80 (Biolegend), 7AAD (BD Biosciences) and anti-mNeuropilin-1 Allophycocyanin conjugated Rat IgG2A (R&D Systems) or Rat IgG2A Isotype Control Allophycocyanin conjugated (R&D Systems).
For analysis of CX3CR1 and CD45 expression, additional extracellular staining was performed using the above mentioned antibodies supplemented with Alexa Fluor 700 anti-mouse CD45.2 (Biolegend) and anti-mouse CX3CR1 Phycoerythrin conjugated Goat IgG (R&D Systems) or Goat IgG Isotype. Control Phycoerythrin conjugated FACS was performed on a LSRII (BD Biosciences) device and data were analysed using FlowJo™ software (version 7.6.5).
BrdU injections. Wild-type mice subjected to OIR or kept in normoxic conditions were injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU; Sigma) at the dose of 1 mg/mouse dissolved in PBS at P13.
Analysis of BrdU incorporation. The staining was performed on the retinal cells from P14 WT mice. Samples were obtained as described above. Extracellular staining was performed as described above (CD45.2 (intermediate/low); Gr-1−; CD11b+, F4/80+; 7AAD). Cells were then fixed with Cytofix/Cytoperm™ Buffer (BD Biosciences) for 30 min and permeabilised with Perm/Wash™ Buffer (BD Biosciences) for 10 min. Next, cells were treated with 300 ug/mL of DNAse for 1 h at 37° C. and washed with Perm/Wash™. Intracellular staining of BrdU was performed using anti-BrdU-PE antibodies (Ebioscience) or mouse IgG1 κ Isotype Control PE conjugated (Ebioscience) for 25 min at 4° C. Cells were then washed in Perm/Wash™ and resuspended in PBS+3% fetal bovine serum before FACS analysis on a LSRII (BD Biosciences).
Vitrectomy. All patients previously diagnosed with PDR were followed and operated by a single vitreoretinal surgeon (FAR). Control patients were undergoing surgical treatment for non-vascular pathology (ERM (epiretinal membrane) or MH (macular hole)) by the same surgeon. In an operating room setting, patients underwent surgery under local retro/peribulbar anesthesia. A 5% povidone-iodine solution was used to clean the periocular skin and topical instillation into the eye and within the cul-de-sac was left in place for 5 minutes. Three-port 25-gauge transconjunctival pars plana vitrectomy was performed through 25-gauge valved cannulas (Alcon). Under microscope visualization using a wide-angle viewing system (Resight™, Zeiss), undiluted vitreous was collected with a 25-gauge vitrector. After vitreous biopsy, the infusion line was opened and vitrectomy and membrane peeling was performed in the usual fashion to treat diabetic vitreous hemorrhage and tractional retinal detachment. This was followed by panretinal endolaser photocoagulation, fluid-air exchange, and intravitreal anti-VEGF injection.
Quantification of SEMA3A protein by ELISA. Vitreous samples were frozen on dry ice and immediately after biopsy and stored at −80°. Samples were centrifuged at 15000×g for 5 minutes at 4° C. prior to analysis. SEMA3A levels were quantified in supernatants using enzyme-linked immunosorbent assays (ELISAs) following manufacturer's instructions (USCN Life Science Inc.).
Assessment of SEMA3A protein levels by Western-blot. Equal volumes of vitreous fluid (20 uL) from PDR and control patients were assessed by standard SDS-PAGE technique for the presence of SEMA3A (Abcam).
Real-time PCR analysis. RNA was isolated using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma) and digested with DNase I to prevent amplification of genomic DNA. Reversed transcription was performed using M-MLV reverse transcriptase (Life Technologies) and gene expression analyzed using Sybr™ Green (BioRad) in an ABI Biosystems Real-Time PCR machine. β-actin was used as a reference gene (see Table 2 in Example 10 for details on the sequence of the oligonucleotides used.
Immunohistochemistry. For visualization of pan-retinal vasculature, flatmount retinas were stained with stained with Rhodamine labeled Griffonia (Bandeiraea) Simplicifolia Lectin I (Vector Laboratories, Inc.) in 1 mM CaCl2 in PBS for retinal vasculature and anti-rat Neuropilin-1 antibody, (goat IgG; R&D Systems) and IBA1 (rabbit polyclonal; Wako).
Primary peritoneal macrophages culture. Adult WT or LyzMcre/NRP1fl/fl mice were anesthetized with 2% isoflurane in oxygen 2 L/min and then euthanized by cervical dislocation. Then, a small incision in abdominal skin of mouse was performed. Skin was pulled to each size of the mouse and peritoneal cavity was washed with 5 ml of PBS plus 3% FBS for 2 min. Then, the harvested cells were centrifuged for 5 min at 1000 rpm, resuspended in medium (DMEM F12 plus 10% FBS and 1% Streptomycin/Penicillin) and plated. After 1 h of culture at 37° C. under a 5% CO2 atmosphere the medium was changed and cells were cultured for the next 24 h in the same conditions before use in cytokine or transwell migration assay.
Transwell migration assay. Migration assays were performed in 24-well plates with 8 μm pore inserts. Primary peritoneal macrophages (5×105 cells) resuspended in 200 μl of medium (DMEM F12 plus 10% FBS and 1% Streptomycin/Penicillin) were added to the upper chamber. 800 μl of medium with or without migratory factors: MCP-1 (100 ng/ml), SEMA3A (100 ng/ml), and VEGF165 (50 ng/ml) was added to the lower chamber. Cells were allowed to migrate through the insert membrane overnight at 37° C. under a 5% CO2 atmosphere. In some experiments, cells were first pretreated with Y-27632 (Sigma), selective ROCK (Rho-associated coiled coil forming protein serine/threonine kinase) inhibitor (100 μg/ml) for 1 h at 37° C. The inserts were then washed with PBS, and nonmigrating cells were swabbed from the upper surface of the insert membrane with cotton buds. Then the membranes with migrated cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes, washed twice with PBS and mounted on the slide. The cells were stained using mounting medium with DAPI (Vector Laboratories, Inc.). Then, 9 random fields per each membrane were photographed using an inverted fluorescence microscope at 20× magnification and the cells were counted using ImageJ software.
Choroidal explants and microvascular sprouting assay. The ex vivo choroid explants and quantification of microvascular sprouting as described previously (56). Briefly, choroids from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice (n=6 for each condition) were dissected shortly after enucleating eyes. After plating segmented choroids into 24 well tissue culture plates and covering with Matrigel™ (BD Biosciences), samples were treated with either EGM™-2 medium, EGM-2 medium with PBS filled liposome (liposome-PBS), or EGM™-2 medium with Dichloromethylenediphosphonic acid disodium salt filled liposome (liposome-Clodronate) (Sigma). The packaging of liposomes was performed according to (57). Twelve hours later, liposomes containing passenger compounds were removed from the wells followed by washing with PBS. Macrophages from primary peritoneal macrophage cultures (from either LysM-Cre/Nrp1+/+ or LysM-Cre/Nrp1fl/fl mice) were added to choroidal explant cultures to investigate the impact of macrophages on microvascular sprouting.
Soluble recombinant NRP1. Wild-type mice subjected to OIR were intravitreally injected with rmNRP1 trap-1 (
Recombinant proteins used. Recombinant mouse CCL2/JE/MCP-1 (from E. coli) (R&D Systems) concentration used in vitro 100 ng/ml. Recombinant human SEMA3A Fc chimera (from murine myeloma cell line, NS0) (R&D Systems) concentration used in vitro 100 ng/ml. -Recombinant human VEGF165 (PeproTech) concentration used in vitro 50 ng/ml.
Statistical analyses. Data are presented as mean±s.e.m. Student's T-test and ANOVA were used, where appropriate, to compare the different groups; a P<0.05 was considered statistically different. For ELISA, statistical analysis was performed using nonparametric Mann-Whitney test (GraphPad Prism).
Study approval: Human samples. We obtained approval of human clinical protocol and informed consent form by Maisonneuve-Rosemont Hospital (HMR) ethics committee (Ref. CER: 10059) and recruitment of patients for local core vitreal biopsy sampling from patients afflicted with T1DM or T2DM. The entire procedure was performed as an outpatient procedure in the minor procedure room within the ambulatory clinic from the Department of Ophthalmology at Maisonneuve-Rosemont Hospital. All instruments were opened and handled in a sterile manner. The study conforms to the tenets of the declaration Helsinki.
Study approval: Animals. All studies were performed according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care Committee of the University of Montreal in agreement with the guidelines established by the Canadian Council on Animal Care. C57131/6 wild-type (WT) were purchased from The Jackson Laboratory. LyzM-Cre (Lyz2tm1(cre)Ifo/J; no. 004781) and Neuropilin 1 floxed mice (Nrp1tm2Ddg/J; no. 005247) were purchased from The Jackson Laboratory.
To determine whether MPs (mononuclear phagocytes) such as microglia or macrophages partake in the vascular pathogenesis associated with proliferative retinopathies, FACS analysis was first carried-out on whole mouse retinas to elucidate the kinetics of macrophage/microglial accumulation throughout the evolution of oxygen-induced retinopathy (OIR,
Importantly, at each time point investigated, we observed a proportional increase in NRP1-positive MPs in OIR with a rise of 37% at P10 (P=0.0240) (
In order to establish the role of MP-resident NRP1 in retinopathy, a myeloid specific knockout of NRP1 was generated by intercrossing Nrp1 floxed mice with LysM-Cre mice (36) yielding LysM-Cre/Nrp1fl/fl progeny. The resulting mice showed an ˜80% decrease in NRP1 expression in retinal MPs when compared to LysM-Cre/Nrp1+/+ littermate controls (P=0.0004) (
Given the pronounced influx of NRP1+ macrophage/microglia during OIR, the localization of these cells during the progression of disease was next determined. Immunofluorescence on retinal flatmounts revealed that NRP1-positive macrophage/microglia (co-labelled with IBA1 and NRP1) were intimately associated with nascent pathological tufts at P14 of OIR (
To establish the clinical relevance of our findings on the obligate role of NRP1 in MP chemotaxis in retinopathy, the concentrations of SEMA3A directly in the vitreous of patients suffering from active PDR was determined. Seventeen samples of undiluted vitreous were obtained from patients suffering from PDR and 17 from control patients with nonvascular pathology. Detailed characteristics of patients are included in Table 1 (Example 1). Control patients (20) presented with non-vascular pathology and showed signs of non-diabetes-related retinal damage such as tractional tension on vasculature (
Consistent with a role in PDR, ELISA-based detection of SEMA3A revealed a 5-fold higher concentrations of the protein in the vitreous humor of patients with PDR when compared to vitreous from control patients (P=0.0132) (
To obtain an accurate kinetic profile of expression of the two prominent ligands of NRP1 in proliferative retinopathy, levels of SEMA3A and VEGF messages in the mouse model of OIR were determined. Real-time quantitative PCR (RT-qPCR) on whole retinas revealed that SEMA3A was robustly induced in OIR both during the hyperoxic (vasodegenerative) phase at P10 and the ischemic/neovascular stage from P12 to P17 (
Next, laser capture micro-dissection (LCM) followed by RT-qPCR was performed on retinal layers in avascular zones to pinpoint the source of SEMA3A and VEGF messages in OIR (
In order to determine if the noted rise in NRP1+ MPs was due to an influx from systemic circulation or an increase in MP proliferation within the retina, the local retinal proliferation of these cells was investigated. Mice were systemically injected with BrdU at P13 (24 hours prior to sacrifice) and FACS analysis was carried out on retinas (
In light of the requirement of NRP1 for myeloid cell mobilization to sites of vascular lesion (
To investigate the impact of NRP1 expressing macrophages on microvascular angiogenesis, choroid tissue from either LysM-Cre/Nrp1+/+ mice or LysM-Cre/Nrp1fl/fl mice was isolated and grew in Matrigel™ to assess microvascular sprouting. Choroids from LysM-Cre/Nrp1fl/fl mice sprout ˜20% less microvessels when compared to ones from LysM-Cre/Nrp1+/+ mice (P=0.018) (
Given the obligate role of NRP1 cell signaling in MP infiltration during the early stages of OIR (
High affinity traps to inhibit/neutralize SEMA3A were generated. These traps were derived from Neuropilin 1 (NRP1) and were optionally coupled to 6×-His tag or FC proteins (see
Methods
Cell culture and material. The human Neuropilin 1 (GenBank™ accession NM_003873, SEQ ID NO: 66) was acquired from Origene Inc. The Origen clone comprises a conservative mutation at amino acid 140 which changes the leucine for an isoleucine. The 293T (ATCC) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The pFUSE-hIgG1-Fc1 vector was purchased from InvivoGen Inc.
Cloning. The extracellular domain of Neuropilin-1 (residues 1-856), or portions of it, were PCR amplified from Origene clone RC217035 using the Phusion™ high fidelity polymerase (New England Biolabs) and cloned in the EcoR1-BgIII of pFUSE-hIgG1-Fc1 in frame with the human FC-1 coding sequence. Constructs coding for the soluble versions of the traps were generated by inserting a sequence coding for a TEV protease cleavage site followed by 6× His residues and a stop codon upstream of the FC coding portion of the corresponding FC constructs. Additional deletions (b1, b1b2) or VEGF165 binding mutations (e.g., Y297A) were introduced using the Q5 site directed mutagenesis kit (NEB). All constructs sequences were verified by Sanger sequencing (Genome Quebec). The nucleotides and amino acid sequences of the assembled traps are depicted in
Evaluation of traps' expression in human cells. Constructs coding for the mouse and human traps were transfected in 293T cells. Cells were grown for 48 hrs post transfection in FreeStyle™ 293 medium (Invitrogen). Cell lysates were prepared from 293T cells 48 hours post-transfections. Cells were extensively washed with PBS and lysed in ice cold lysis buffer (50 mM HEPES pH7.5, 150 mM NaCL, 1.5 mM MgCl2, 1% Triton X-100 and 10% glycerol) supplemented with standard amounts of protease inhibitors (AEBSF, TPCK, TLCK, aprotinin, leupeptin, pepstatin and E64, Sigma). Cell lysates were cleared by micro centrifugation (12000 g, 20 minutes). Lysates concentrations were determined by standard micro BCA (Sigma). Equal amounts of protein were loaded on 5-20% PAGE-SDS gradient gels and transfered to PVDF (Amersham). Cleared conditioned media from transfected cells were incubated with either Protein A sepharose (Pharmacia) or Talon resin (Clontech) for FC or 6×His tag. Resins were washed with PBS and diluted in 2× PAGE-SDS sample buffer prior to gel separation and transfer. The antibody used in immunoblottings were the anti-human Neuropilin-1 (Cell signaling), the mouse monoclonal anti-6×-HIS (In Vitrogen) and the reporter HRP linked anti-human, mouse and rabbit IgG (BioRAD). All antibodies were used at a 1/2000 dilution. Chemiluminescent signal was captured using a Fuji imaging system after incubation of membranes with ECL (Amersham).
Traps expression and purification. 293-T cells were transfected with plasmids encoding the various traps by either the Polyethylamine (PEI) or the calcium phosphate precipitation standard transfections methods. The next day cells were washed twice with serum free media and fed with serum free complete media (Free style 293 media, InVitrogen). Conditioned medium were collected after 60-72 hrs of growth in serum free media and cleared from cellular debris by swing bucket centrifugation (2000 RPM, 20 minutes). FC traps were purified from conditioned media of transfected 293T cells by passage on Protein A or G sepharose (Pharmacia) followed by extensive washes with PBS and elutions with 0.1 M glycine pH 3.0. Elution fractions were neutralised immediately by the addition of 1/10 volume 1 M Tris pH 8 and 1/10 volume of 10× PBS pH 7.4. Soluble 6× HIS tagged traps were purified from conditioned media of transfected 293T cell by passage on Talon agarose (Clontech) followed by extensive washes with PBS and stepwise imidazole elutions (Range 10-150 uM typically). Samples of purification fractions of traps were analysed on 5-15% or 5-20% gradient PAGE-SDS gels. Gel were stained using the Safely Blue staining kit (InVitrogen).
Sterile formulation of purified traps for in vivo injections. Purifications elution fractions from 40 ml of conditioned media were pooled and diluted to a total volume of 10 ml in PBS. Diluted trap proteins were sterilized by filtration through a 0.2 uM low protein binding filter (Progene). Protein solutions were concentrated and buffer exchanged with PBS on sterile PES concentration devices (Pierce, nominal MWCO 30 KD). Sterile concentrated Traps samples (˜30-50 ul) were analysed and stained on PAGE-SDS as described above.
Example 11 Affinity of Traps for SEMA3A and VEGFProduction of AP-VEGF165. the coding sequence of the human VEGF165 variant 1 (NM_001025366) was sub-cloned in the pAPtag5 vector (GenHunter), in-frame with an Alkaline Phosphatase domain (AP-VEGF165). HEK293T cells were transfected with the AP-VEGF165 construct using a polyethylenimine (PEI) transfection method. Following the overnight transfection step, cells were cultured for an additional 60 hr in serum free media (Invitrogen). The cell media were collected and concentrated on a PES device (Pierce). The concentrated AP-VEGF165 ligand was analysed on PAGE-SDS and quantified using SimplyBlue safe stain (Life technologies).
Sema 3A and AP-VEGF165 binding assays. Saturation curves for the determinations of KD of binding to SEMA 3A or VEGF165 were obtained as follow. Wells of high protein binding 96 well plates (Maxisorp, Nunc) were coated with purified traps diluted in PBS and blocked afterward with binding buffer (PBS containing 2% casein and 0.05% Tween 20). The SEMA3A-FC (R&D systems) or AP-VEGF165 ligands were diluted in binding buffer over an extensive range of concentrations and added to wells. Following an overnight incubation, wells were washed with PBS containing 0.05% tween. Bound SEMA3a-FC was detected using an HRP-linked anti-Human IgG (Biorad) and ECL substrate (Pierce). Alternatively, bound AP-VEGF165 was detected using CPD star substrate (Roche). The Chemiluminescent signal was acquired on a TECAN reader. Dissociation constant (KD) were determined by non-linear curve fitting using the Graph Pad prism software.
The relative affinity of traps of the present invention to SEMA3A and VEGF has been assessed. Traps were prepared as described in Example 10. Schematic representation of traps tested is also provided in
Soluble NRP1 traps of the present invention bind more efficiently to SEMA3A than VEGF. Such preference for SEMA3A was found surprising since SEMA3A and VEGF are considered to normally have the same general affinity for NRP1. Increased affinity for SEMA3A may be advantageous in conditions where SEMA3A inhibition is preferred over inhibition of VEGF and may reduce side effects associated with VEGF inhibition.
Example 12 Therapeutic Intravitreal Administration of Soluble NRP1 Reduces MP Infiltration and Pathological Neovascularization in RetinopathyTo determine the translational potential of the above findings, a soluble recombinant mouse (rm)NRP1 mTrap 1 polypeptide (
Mouse model of sepsis. Studies were performed according to the regulations from the Canadian Guidelines for the Use of Animals in Research by the Canadian Council on Animal Care. LPS injections were delivered intra-peritoneally (i.p) in 6-8 weeks old C57BL/6 mice.
Survival assay. For generation of survival data, mice were challenged with a single intraperitoneal injection of LPS at 25 mg/kg, in a volume of nearly 100 ul adjusted to mouse weight. Mice were then monitored until reaching critical limit points defined by the Canadian Council of Animal Care.
Measurement of pro-inflammatory cytokines. For assessment of pro-inflammatory cytokines, mice were challenged i.p. with a single intraperitoneal injection of LPS at 15 mg/kg and sacrificed at various time points up to 24 hours. Tissues (Brain, Liver, Kidney) were removed and mRNA was isolated using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma) and digested with DNase I to prevent amplification of genomic DNA. Reversed transcription was performed using M-MLV reverse transcriptase and gene expression analyzed using SybrGreen in an ABI Biosystems Real-Time PCR machine. β-actin was used as a reference gene.
Primary peritoneal macrophages culture. Adult WT or LyzMcre/NRP1fl/fl mice were anesthetized with 2% isoflurane in oxygen 2 L/min and then euthanized by cervical dislocation. Then, a small incision in abdominal skin of mouse was performed. Skin was pulled to each size of the mouse and peritoneal cavity was washed with 5 ml of PBS plus 3% FBS for 2 min. Then, the harvested cells were centrifuged for 5 min at 1000 rpm, resuspended in medium (DMEM F12 plus 10% FBS and 1% Streptomycin/Penicillin) and plated. After 1 h of culture at 37° C. under a 5% CO2 atmosphere the medium was changed.
Cytometric Bead Array (CBA). CBA was performed according to manufacturer's guidelines (BD Bioscience). Macrophages were isolated from wild type or LyzMcre/NRP1fl/fl mice and subjected to SEMA3A (100 ng/ml) or vehicle for 12 hours and processed by CBA.
Trap and anti-VEGF antibody administration. Mice experimental model of sepsis were treated with human or mice NRP1 trap-1 (
Experimental design: 3 mice per group. Groups: 1-Vehicle, 2-LPS, and 3-LPS+NRP1 Trap 1-Vehicle: NaCl, 2-LPS: 15 mg/kg; and 3-LPS+NRP1-trap: Mice received i.v. a single injection of 4 ug (in a volume of 100 uL) of recombinant mouse NRP1-trap corresponding to 0.2 mg/kg, few minutes after LPS injection.
Permeability tests. For permeability assays, mice were challenged i.p. with a single intraperitoneal injection of LPS at 15 mg/kg, and sacrificed 24 hrs later for tissue sampling. Changes in liver, kidney, and brain vascular permeability were assessed by quantifying Evans Blue (EB) extravasation in tissue. After 24 hrs, a solution of 10 mg/ml of EB was injected intravenously (55 mg/kg). Two hours later, mice were sacrificed and perfused through the heart with PBS. Tissues were then removed, allowed to dry at room temperature 24 hrs, and dry weights were determined. EB was extracted in formamide overnight at 65° C. EB was then measured at 620 and 740 nm in spectrophotometer.
Real-time PCR analysis. RNA was isolated using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma) and digested with DNase I to prevent amplification of genomic DNA. Reversed transcription was performed using M-MLV reverse transcriptase (Life Technologies) and gene expression analyzed using SybrGreen (BioRad) in an ABI Biosystems Real-Time PCR machine. β-actin was used as a reference gene. See Table 3 below for details on the sequence of the oligonucleotides used.
Given the link between SEMA3A, NRP1 and the innate immune response in OIR (as demonstrated in Examples 2-9 above), the implication of the NRP1-dependent cellular response in general systemic inflammation was next assessed. This was first explored by determining the kinetics of SEMA3A expression during septic shock.
LPS was administrated (15 mg/kg) to 6-8 weeks old C57BL/6 mice (n=5) and mice were sacrificed at 0, 4, 8, 12 and 24 hours following LPS administration. Key organs such as brain, kidney, lung and liver were collected and mRNA isolated. Levels of SEMA3A mRNA were robustly induced in all organs analyzed as soon as 6 hours after LPS injection and persisted for 24 hours (
Given the contribution of monocytes and myeloid cells to the acute inflammatory response and the presence of NRP1 on myeloid cells, the contribution of SEMA3A and myeloid-resident NRP1 in the production of inflammatory cytokines was determined.
Isolated macrophages were exposed to SEMA3A (100 ng/ml) or vehicle and the production of cytokines was analyzed by Cytometric Bead Array (CBA). Results presented in
Because myeloid-resident NRP1 was important for the release of pro-inflammatory cytokines such as IL-6 and TNF-α in vitro, its contribution was next explored in vivo. LyzM/NRP1fl/fl and control wild-type mice were administered vehicle or LPS (15 mg/kg) and brains and livers were collected 6 hours post LPS injection. Real-time PCR analysis of TNF-α (
One of the pathological features of severe septic shock though to contribute to organ failure is the compromise of blood barriers (blood and air in lung, blood and urine in the kidney, blood and bile in liver, and humoral molecules in the brain). Given a role for SEMA3A in the breakdown of the blood retinal barrier (46) and the present novel data on the expression of SEMA3A during sepsis, the effect of neutralizing SEMA3A with a trap derived from the extracellular domain of human NRP1 was assessed (Trap-1, without FC,
To determine the therapeutic benefits of neutralization of NRP1 ligands or NRP1 inhibition during sepsis, survival studies were performed. A high dose of LPS (25 mg/kg) was administered to mice. Mice were then monitored, and ethically sacrificed, when appropriate endpoints were achieved. In the second group, mice were injected i.v. with 4 ug of recombinant Trap-1 without FC (0.2 mg/kg,
Similar results were obtained with mice harboring a specific knock out of NPR1 in myeloid cells (
Taken together, these results highlight the therapeutic value of inhibiting NRP1-dependent cell signaling in sepsis treatment.
Example 19 NRP1-Derived Trap Lowers Production of Inflammatory Cytokines in Septic ShockGiven the therapeutic benefit of NRP1-trap on survival rates in septic shock, the impact of neutralization of NRP1 ligands on production of inflammatory cytokines during septic shock was next determined. Wild-type mice were administered i) vehicle (n=3); ii) LPS (15 mg/kg) (n=3) or iii) LPS and NRP1 mouse Trap 1 (without FC,
The mice used in this study were 2- to 3-month old male C57Bl/6 mice (22-28 g).
MCAO model. MCAO mouse model was performed using the intraluminal suture technique described by Rousselet et al. (66). Briefly, mice were anesthetized in a chamber with 3% isoflurane in oxygen (1 L/min) and analgesized with buprenorphine (0.1 mg/kg body weight subcutaneously). Anesthesia was maintained during the operation using 1.5% isoflurane in oxygen provided via a face mask. The rectal temperature was recorded and kept stable at 37±0.5° C. with a heating pad. After a midline incision at the neck, the right carotid bifurcation was exposed and the common carotid artery (CCA) was temporarily occluded using 5-0 silk suture. The bifurcation of the right internal common carotid artery (ICA) and external common carotid artery (ECA) was separated. A permanent suture was placed around the ECA, as distally as possible, and another temporary suture slightly tight was placed on the ECA distal to the bifurcation. The right ICA was temporarily occluded with 5-0 silk suture to avoid bleeding. Then, a small hole in the ECA was cut between permanent and temporary sutures through which a 12 mm-long 6-0 silicon-coated (about 9-10 mm was coated with silicon) monofilament suture was introduced. The filament was advanced from the ECA into the lumen of the ICA until it blocked the origin of the middle cerebral artery (MCA) in the circle of Willis. Sham animals were obtained by inserting the monofilament into the CCA, but without advancing it to the MCA. The suture on the ECA was tightly tied to fix the monofilament in position. Thirty minutes after MCAO, the monofilament was completely removed to allow reperfusion. The temporary suture on the CCA was also removed to allow blood recirculation. After the wound was closed, 1 ml of saline solution was injected subcutaneously to avoid postsurgical dehydration. The mouse was placed in a cage and kept on the heating pad for 1 h. Meantime, when the mouse was fully awake from anesthesia, it was checked for some basic motor deficits (circling while walking and bending while hold by tail; indicators of the success of the operation) and NRP1-Trap-1 without FC (
Determination of infarct volume. Following neurological evaluation (see section below) performed 24 h after MCAO the animals were deeply anesthetized with 3% isoflurane in oxygen (1 L/min) and decapitated. The brains were immediately isolated and transferred into isopentane cooled on dry ice and then stored at ˜80° C. Then, the frozen brains were coronally cut into 20-μm sections in a cryostat at −22° C. and every 15th slice was mounted on positively charged glass slides. Cerebral sections were stained with cresyl violet for 15 min. Each section was photographed. The areas of infarction were delineated on the basis of the relative lack of staining in the ischemic region and measured by using NIH ImageJ software. Infarct area in each section was determined as the total area of the contralateral hemisphere minus the non-affected area of the ipsilateral hemisphere.
Neurological evaluation. One hour after operation, as well as 24 h after MCAO, animals were subjected to a series of motor tests performed. The examinations and scoring were as follows: 0, Normal; 1, Contralateral front or rear limb flexion upon lifting of the whole animal by the tail; 2, Circling to the contralateral side while walking and C-shaped lateral bending while hold by tail; 4 Circling to the contralateral side while walking and C-shaped lateral bending while hold by tail with limb flexion; 5, Comatose or moribund. The magnitude of the obtained neuroscore is directly proportional to the severity of impairment.
Example 21 NRP1-Trap Protects Against Cerebral Ischemia and StrokeIn order to assess the outcome of SEMA3A neutralization on cerebral ischemia or stroke, adult (8-12 week-old) mice were subjected to the transient middle cerebral artery occlusion (MCAO) model. Experimental details are provided in Example 20. Briefly, following termination of MCAO, mouse NRP1-trap (Trap-1, without FC), 0.4 ug in 125 ul of PBS, (
Neurological impairment was assessed by neurological scoring of the presence of limb flexion, C-shaped lateral bending of the body and circling movements. MCAO mice that were not showing circling and bending behaviour 1 hour after operation were excluded from the further study (
Taken together, these results show that inhibition of the NRP1 pathway protects against cerebral ischemia and stroke and reduce the neurological impairment associated with cerebral ischemia and stroke.
Example 22 Neuropilin-Derived Traps Enhance Vascular Regeneration and Prevent Pathological Neovascularization in Ischemic Retinas in Mouse Model of Diabetic Retinopathy and Retinopathy of PrematurityPathological vascular degeneration as well as pre-retinal vascular proliferation were studied using the well-established mouse model of oxygen-induced proliferative retinopathy (OIR)(Smith et al., 1994). This model is based on retinopathy of prematurity (ROP) and is regularly used as a proxy for the proliferative (angiogenic phase) of diabetic retinopathy and ROP.
Nursing mothers and their pups were exposed to 75% oxygen from P7-P12. Both vaso-degenerative (assessed at P12) and vaso-proliferative (assessed at P17) phases are present and are highly reproducible making evaluation of interventions on disease progression accurate and swift. Trap G (SEQ ID NO: 38), or Trap M (lacking the b2 and c domains, SEQ ID NO: 42), was injected into the vitreous at P12 (1 ul at 0.5 ug/ul). Dissected retinas were flatmounted and incubated overnight with fluoresceinated isolectin B4 (1:100) in 1 mM CaCl2 to determine extent of avascular area or neovascularization area at P17. Avascular areas were determined in lectin stained retinas as zones devoid of staining. Neovascularization was determined as areas of saturated lectin staining which demarcates pre-retinal tufts (54, 55).
Trap G, was shown to effectively enhance vascular regeneration by over 40% when compared to vehicle control (
Neuropilin-Derived Traps Decrease Vascular Leakage in Diabetic Retinas.
The influence of Traps on vascular leakage/permeability in diabetic retinopathy was also studied in the streptozotocin (STZ) model of Type 1 diabetes. STZ (55 mg/kg) was administered over 5 consecutive days to ˜6 week-old C57BL/6J mice and glycemia was monitored. Mice were considered diabetic if their non-fasted glycemia was higher than 17 mM (300 mg/dL). Mice were administered intravitreally with 0.5 ug (0.5 ug/ul) of Trap G (SEQ ID NO: 38) or M (SEQ ID NO: 42) or with mouse anti-VEGF antibody (AF-493-NA, from R&D) at 6 and 7 weeks after STZ administration. Alternatively, mice were injected intravitreally at 12 and 13 weeks post STZ and vascular permeability assessed at 14 weeks. Mice were hyperglycemic/diabetic at least 3 weeks prior to intravitreal injections with SEMA traps (see
Both Trap-G (SEQ ID NO: 38) and Trap-M (SEQ ID NO: 42) significantly reduced vascular permeability by over 40% (
The effect of NRP1 trap G (SEQ ID NO: 38) on choroidal neovascularization (CNV) was determined in a mouse model of age-related macular degeneration (AMD). To induce CNV and thus mimic wet AMD in mice, laser coagulations on 6-8 week old mice (1-2 disc diameters) were performed from the papillae using an Argon laser (532 nm) mounted on a Coherent slit lamp (400 mW, 50 ms and 50 μm) (Combadiere et al., 2007). Following laser burn, treated mice were injected intravitreally with 0.5 ug of Trap G. Fourteen days (P14) later, choroids were radially incised, flat-mounted and stained with the endothelial cell marker fluoresceinated Isolectin B4 (animals were also optionally perfused with fluorescein dextran to visualize luminal vessels) and volumes of CNV were measured by scanning laser confocal microscopy (Takeda et al., 2009).
Trap G was shown to significantly reduce choroidal neovascularization at day 14 post laser-burn (
The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
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Claims
1. An ophthalmic pharmaceutical composition comprising (a) a soluble neuropilin-1 (NRP1) polypeptide in an amount suitable to reduce eye inflammation in a subject, the NRP1 polypeptide comprising an amino acid sequence having at least 90% identity with residues 22-424 of SEQ ID NO: 107, wherein said NRP1 polypeptide does not comprise the full b2 and c domains of native human NRP1; and (b) at least one carrier for ophthalmic administration.
2. The composition of claim 1, wherein said NRP1 polypeptide comprises an amino acid sequence having at least 95% identity with residues 22-424 of SEQ ID NO: 107.
3. The composition of claim 1, wherein said NRP1 polypeptide comprises residues 22-424 of SEQ ID NO: 107.
4. The composition of claim 1, wherein said at least one carrier is an aqueous or oily carrier.
5. The composition of claim 1, wherein said at least one carrier is a physiological saline buffer.
6. The composition of claim 1, wherein said composition is a suspension, solution or emulsion.
7. The composition of claim 1, which is formulated for ocular administration.
8. The composition of claim 1, wherein said composition is an eye drop formulation.
9. The composition of claim 1, wherein said composition is an injectable formulation.
10. The composition of claim 1, wherein said composition further comprises a preservative.
11. The composition of claim 9, wherein said composition is present in an injection device, an ampoule or a multi-dose container.
12. A method for preparing the ophthalmic pharmaceutical composition of claim 1 comprising mixing: (a) the soluble NRP1 polypeptide comprising an amino acid sequence having at least 90% identity with residues 22-424 of SEQ ID NO: 107, wherein said NRP1 polypeptide does not comprise the full b2 and c domains of native human NRP1; with (b) the at least one carrier for ophthalmic administration.
13. The method of claim 12, wherein the composition is formulated for ocular administration.
14. A method for reducing ocular inflammation in a subject, comprising administering in the eye of the subject an effective amount of the composition of claim 1.
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| WO 03/035100 | May 2003 | WO |
| WO 2007/009071 | January 2007 | WO |
| WO 2008/143666 | November 2008 | WO |
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Type: Grant
Filed: Aug 3, 2020
Date of Patent: May 16, 2023
Patent Publication Number: 20210061915
Assignee: RSEM, LIMITED PARTNERSHIP (Montreal)
Inventors: Przemyslaw Sapieha (Montréal), Normand Beaulieu (Montréal)
Primary Examiner: Aurora M Fontainhas
Application Number: 16/983,185
International Classification: A61K 47/68 (20170101); C12N 15/62 (20060101); C07K 16/28 (20060101); A61K 31/713 (20060101); A61K 31/7105 (20060101); C07K 14/71 (20060101); C07K 16/22 (20060101); A61K 39/395 (20060101); C12N 15/113 (20100101); A61K 39/00 (20060101); A61K 38/00 (20060101);
